A Heritable Recombination system for synthetic Darwinian evolution in yeast.

Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology.

Rapid chemoselective bioconjugation through oxidative coupling of anilines and aminophenols.

A highly efficient protein bioconjugation method is described involving addition of anilines to o-aminophenols in the presence of sodium periodate. The reaction takes place in aqueous buffer at pH 6.5 and can reach high conversion in 2-5 min. The major product was characterized using X-ray crystallography, which revealed that an unprecedented oxidative ring contraction occurs after the coupling step. The compatibility of the reaction with protein substrates has been demonstrated through attachment of small molecules, polymer chains, and peptides to p-aminophenylalanine residues introduced into viral capsids through amber stop codon suppression. Coupling of anilines to o-aminophenol groups derived from tyrosine residues is also described. The compatibility of this method with thiol modification chemistry is shown through attachment of a near-IR fluorescent chromophore to cysteine residues inside the viral capsid shells, followed by attachment of integrin-targeting RGD peptides to anilines on the exterior surface.

Multivalent, high-relaxivity MRI contrast agents using rigid cysteine-reactive gadolinium complexes.

MRI contrast agents providing very high relaxivity values can be obtained through the attachment of multiple gadolinium(III) complexes to the interior surfaces of genome-free viral capsids. In previous studies, the contrast enhancement was predicted to depend on the rigidity of the linker attaching the MRI agents to the protein surface. To test this hypothesis, a new set of Gd-hydroxypyridonate based MRI agents was prepared and attached to genetically introduced cysteine residues through flexible and rigid linkers. Greater contrast enhancements were seen for MRI agents that were attached via rigid linkers, validating the design concept and outlining a path for future improvements of nanoscale MRI contrast agents.

Genome-free viral capsids as carriers for positron emission tomography radiolabels.

PURPOSE: We have developed a modular synthetic strategy to append imaging agents to a viral capsid. PROCEDURES: The hollow protein shell of bacteriophage MS2 (mtMS2) was labeled on its inside surface with [18F]fluorobenzaldehyde through a multistep bioconjugation strategy. An aldehyde functional group was first attached to interior tyrosine residues through a diazonium coupling reaction. The aldehyde was further elaborated to an alkoxyamine functional group, which was then condensed with n.c.a. [18F]fluorobenzaldehyde. Biodistribution of the radioactive MS2 conjugates was subsequently evaluated in Sprague-Dawley rats. RESULTS: Relative to fluorobenzaldehyde, fluorine-18-labeled MS2 exhibited prolonged blood circulation time and a significantly altered excretion profile. It was also observed that additional small molecule cargo installed inside the capsids did not alter the biodistribution. CONCLUSIONS: These studies provide further insight into the pharmacokinetic behavior of nanomaterials and serve as a platform for the future development of targeted imaging and therapeutic agents based on mtMS2.

Oxidative coupling of peptides to a virus capsid containing unnatural amino acids.

This Communication describes the chemo- and site-selective coupling of cell type-specific targeting peptides to a virus capsid containing aminophenylalanine residues.

Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua.

Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to beta-amyrin synthases (EC 5.4.99.-), sharing amino acid sequence identities of up to 86%. Expression of the gene, designated AaBAS, in Saccharomyces cerevisiae, followed by GC/MS analysis, confirmed the encoded enzyme as a beta-amyrin synthase. Through engineering the sterol pathway in S. cerevisiae, we explore strategies for increasing triterpene production, using AaBAS as a test case. By manipulation of two key enzymes in the pathway, 3-hydroxy-3-methylglutaryl-CoA reductase and lanosterol synthase, we have improved beta-amyrin production by 50%, achieving levels of 6 mg.L(-1) culture. As we have observed a 12-fold increase in squalene levels, it appears that this strain has the capacity for even higher beta-amyrin production. Options for further engineering efforts are explored.

Attachment of peptide building blocks to proteins through tyrosine bioconjugation.

Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannich-type strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues in length with anilines at either the N- or C-termini. In the presence of 20 mM formaldehyde, these functional groups were coupled to tyrosine residues on proteins under mild reaction conditions. The identities of the resulting bioconjugates were confirmed using mass spectrometry and immunoblot analysis. Screening experiments have demonstrated that the method is compatible with substrates containing all of the amino acids, including lysine and cysteine residues. Importantly, tyrosine residues on proteins exhibit much faster reaction rates, allowing short peptides containing this residue to be coupled without cross reactions.

Dual-surface-modified bacteriophage MS2 as an ideal scaffold for a viral capsid-based drug delivery system.

With the development of covalent modification strategies for viral capsids comes the ability to convert them into modular carrier systems for drug molecules and imaging agents. With this overall goal in mind, we have used two orthogonal modification strategies to decorate the exterior surface of genome-free MS2 capsids with PEG chains, while installing 50-70 copies of a fluorescent dye inside as a drug cargo mimic. Despite the very high levels of modification, the capsids remained in the assembled state, as determined by TEM, size-exclusion chromatography, and dynamic light scattering analysis. The ability of the polymer coating to block the access of polyclonal antibodies to the capsid surface was probed using a sandwich ELISA, which indicated a 90% reduction in binding. Further experiments indicated that biotin groups placed at the distal ends of the polymer chains were still capable of binding to streptavidin, despite their proximity to the PEG layer. Finally, a modular strategy was developed for the attachment of small-molecule targeting groups to the polymer chains through an efficient oxime formation reaction. As a result of these studies, a robust and versatile new platform has emerged for the potential delivery of therapeutic cargo.